1. Field
This disclosure is concerned generally with a method of purifying proteins and specifically with a three step method involving pH adjustments to purify the protein without the use of precipitation agents or salt solutions.
2. Prior Art
Current methods for purification of proteins include using precipitation agents such as salts or non-salt substances such as polyethylene glycol (PEG). Unfortunately, in many cases the protein yields are less than desired. The methods are often time consuming and often require the use of specialized equipment such as large centrifuges. Also, it is often difficult to scale up a precipitation method and even if this can be done dissolution of the resulting precipitate can be slow and is not always complete.
Another common method for purifying proteins includes passing a protein-containing solution over or through an appropriate ion exchange material at a solution pH which facilitates binding of the desired protein. This is commonly followed by a washing step and an elution step at a different ionic strength or pH which facilitates the release of the protein. See, for example Lamy, J. et al, Arch Biochem. Biophys. 193 pp 140-149 (1979), showing that protein can be eluted from DEAE Sepharose.RTM. by using a descending pH gradient. In general, altering the pH of a protein towards its isoelectric point causes it to lose net charge and elute from an ion exchanger. Elution of materials from ion exchangers by pH change and ionic strength change is described more fully in Morris et al, Separation Methods in Biochemistry Pittman and Sons, London, 1964. See also Robert Scopes, Protein Purification, Springer-Verlag, New York, N.Y., 1982. At page 85, the author points out that the use of a change in buffer pH is generally not very successful (for protein purification).
We have now found that by slightly modifying the above ion exchange/pH method, we can purify a protein such as a monoclonal antibody in a relatively simple manner that avoids protein loss commonly associated with existing protein purification methods, especially methods based on the use of solutions of increasing ionic strength. Details of our method are described below.